JP2014519607A - Protein detection method and detection apparatus - Google Patents

Abstract

A protein detection apparatus and method. The apparatus has a main channel to which either a rail molecule or a biomolecule motor that moves on the rail molecule in a direction corresponding to the polarity of the rail molecule is attached. The subchannel intersects the main channel to receive an analyte containing tau protein. The method includes supplying a specimen to the apparatus and the percentage of altered tau protein contained in the specimen based on the movement state of the biomolecule motor on the rail molecule or the movement state of the rail molecule moved by the biomolecule motor. Detecting.
[Selection] Figure 1

Description

  The present invention relates to a protein detection method and a detection apparatus used in the method.

  Conventionally, kinesin, which is a kind of biomolecular motor, is known to move on a microtubule, which is a kind of rail molecule, with the hydrolysis of ATP (adenosine triphosphate). (For example, refer to Patent Document 1). In this case, the microtubule is a filament having a cylindrical structure having a diameter of about 25 [nm] and a length of several tens [[mu] m] obtained by polymerizing the monomer tubulin. The kinesin moves from the minus end side of the microtubule toward the plus end side.

  In addition, MAP (Microtubule Associated Protein) exists in the microtubule in the living body, and in particular, MAP called tau protein (Tau-Protein) adheres to the microtubule of the brain nerve cell. A normal tau protein (Healthy Tau) contributes to tubulin polymerization and microtubule stability, and supports intracellular substance transport (see, for example, Non-Patent Document 1). However, abnormal tau proteins (or degenerated tau) such as hyperphosphorylated and localized tau proteins (or altered tau proteins) inhibit biomolecular motor movement in vivo. To do.

  In fields such as brain science and neuropathology, altered tau protein forms aggregates (Tangled Tau, ie, Tangles) in the olfactory intestine of the brain, thereby reducing neurological function and resulting memory impairment. It is known that a tau protein abnormal disease (Tauopathy: Tauopathy) represented by Alzheimer's disease occurs (for example, refer nonpatent literature 2). Thus, as a technique for detecting the presence of tau protein on-chip, a method using surface plasmon or a method by immunoassay has been proposed (see, for example, Non-Patent Document 3).

JP 2006-321211 A P. Tolnay, “Tau protein pathology in Alzheimer's disease and related disorders”, Neuropathology and applied neurobiology, vol. 25 (3), pp. 171-187 (1999) T. Kimura et al., EMBO J., 26, 5143-5152, 2007 M. Vestergaard et al., Talanta, 74, 1038-1042, 2008

  However, the conventional technique detects only the presence of tau protein, and cannot detect the presence of altered tau protein such as hyperphosphorylated tau protein or localized tau protein. If tau protein abnormal disease can be diagnosed at a stage where the proportion of altered tau protein is low, it can be treated early by, for example, restoring its function as a normal tau protein using a phosphorylase inhibitor or the like. Is considered possible.

  The present invention solves the above-mentioned conventional problems, and observes the relative movement state of a biomolecular motor with respect to a rail molecule stabilized by paclitaxel, whereby the altered tau protein contained in the specimen is observed. It is an object of the present invention to provide a protein detection method and detection apparatus capable of easily and reliably detecting the ratio.

  Therefore, in the protein detection method of the present invention, a relative movement between a rail molecule having a polarity and a biomolecule motor moving in a direction corresponding to the polarity and a biomolecule motor moving on the rail molecule is provided. A method for detecting a protein based on a state, wherein a rail molecule is stabilized by a stabilizer, either the rail molecule or the biomolecule motor is attached on a substrate, and a specimen containing tau protein is added to the rail. Based on the movement state of the biomolecule motor that is supplied to the molecule and moves on the rail molecule or the movement state of the rail molecule that is moved by the biomolecule motor, the ratio of the altered tau protein contained in the specimen is detected. .

  In another method for detecting a protein of the present invention, the movement of a biomolecular motor on a rail molecule stabilized by a stabilizer is inhibited by a normal tau protein contained in the specimen, and is altered by a modified tau protein. Based on the fact that is not inhibited, the proportion of altered tau protein contained in the specimen is detected.

  In still another protein detection method of the present invention, the stabilizer is paclitaxel or docetaxel.

  In still another method for detecting a protein of the present invention, the movement state may be the movement speed, movement distance or density or number of a biomolecule motor moving on the rail molecule, or moved by the biomolecule motor. The moving speed, moving distance, density or number of rail molecules to be generated.

  In still another protein detection method of the present invention, a microsphere having a biomolecule motor joined to a rail molecule attached on a substrate is further supplied, and the rail is based on the movement state of the microsphere. The movement state of the biomolecule motor moving on the molecule is determined.

  In still another protein detection method of the present invention, a rail molecule having fluorescence is further supplied to a biomolecule motor attached on a base material, and the biomolecule motor is based on a movement state of the rail molecule. The movement state of the rail molecule moved by is determined.

  In the detection device of the present invention, the detection is performed based on the relative movement state of the rail molecule having a polarity and the biomolecule motor moving in the direction corresponding to the polarity and the biomolecule motor moving on the rail molecule. A detection device used in a protein detection method, which extends in a direction intersecting with the main channel, the main channel having the base surface of the base material to which the rail molecules stabilized by the stabilizer are attached, A subchannel to which a specimen containing tau protein is supplied, and the movement state of the biomolecule motor moving on the rail molecule can be observed.

  In another detection apparatus of the present invention, the subchannel is plural, and it is possible to simultaneously observe the influence of the tau protein contained in the plurality of samples on the movement state of the biomolecule motor moving on the rail molecule. is there.

  In still another detection device of the present invention, a microsphere having a biomolecule motor joined thereto is supplied to the main channel, and the biomolecule motor moves on the rail molecule based on the movement state of the microsphere. Is determined.

  In still another detection device of the present invention, the relative movement state between the rail molecule having a polarity and the biomolecule motor moving in the direction corresponding to the polarity and the biomolecule motor moving on the rail molecule is set. A detection device used in a protein detection method based on a nanochannel portion including a plurality of nanochannels having a bottom surface as a surface of a base material to which the biomolecule motor is attached, and one end of the nanochannel portion An inlet part to which rail molecules connected and stabilized by a stabilizer are supplied, and an outlet part connected to the other end of the nanochannel part, which is supplied to the inlet part and passes through the nanochannel part And an outlet portion through which the discharged rail molecules can be observed.

  In still another detection device of the present invention, the rail molecules supplied to the inlet portion have fluorescence, and the outlet portion can detect fluorescence intensity.

  According to the present invention, in the protein detection method, the rail molecule is stabilized by the stabilizer, either the rail molecule or the biomolecule motor is attached to the substrate, and the specimen containing the tau protein is added to the rail molecule. The ratio of the altered tau protein contained in the specimen is detected based on the movement state of the biomolecule motor moving on the rail molecule or the movement state of the rail molecule moved by the biomolecule motor. This makes it possible to easily and reliably detect the proportion of altered tau protein contained in the specimen in vitro.

  Further, in the detection apparatus, a main channel having a base surface on which a rail molecule stabilized by a stabilizer is attached as a bottom surface, and a specimen including a tau protein extending in a direction intersecting the main channel are supplied. It is possible to observe the movement state of the biomolecule motor that moves on the rail molecule. Thereby, although it is a simple structure, the ratio of the altered tau protein contained in the specimen can be easily and reliably detected in vitro.

  Further, in the detection device, a nanochannel portion including a plurality of nanochannels whose bottom surface is a surface of a base material to which a biomolecule motor is attached, and rail molecules connected to one end of the nanochannel portion and stabilized by paclitaxel And an outlet portion connected to the other end of the nanochannel portion, the outlet portion being supplied to the inlet portion and capable of observing the rail molecules discharged through the nanochannel portion. Part. Thereby, although it is a simple structure, the ratio of the altered tau protein contained in the specimen can be easily and reliably detected in vitro.

It is a figure explaining the method to detect the altered tau protein using the 1st detection apparatus in the 1st Embodiment of this invention. It is a schematic diagram which shows the microtubule and kinesin in the 1st Embodiment of this invention. It is a schematic diagram which shows the relationship between the microtubule and the tau protein in the living body in the 1st Embodiment of this invention. It is a conceptual diagram of the bead assay in the 1st Embodiment of this invention. It is a conceptual diagram of the method to detect the altered tau protein in the 1st Embodiment of this invention. It is the microscope picture which image | photographed the bead in the 1st Embodiment of this invention. It is a figure explaining the device used in the experiment in the 1st Embodiment of this invention. It is a figure which shows the influence of tau protein on the motion of kinesin in the 1st Embodiment of this invention. It is a figure which shows the influence of the density of the tau protein which acts on the movement of kinesin in the 1st Embodiment of this invention. It is a figure which shows the influence of incubation time on the motion of kinesin in the 1st Embodiment of this invention. It is a figure which shows the influence of the incubation time on the number of the molecules of kinesin in the 1st Embodiment of this invention. It is a figure which shows the influence of the state of a microtubule on the motion of kinesin in the 1st Embodiment of this invention. It is a 2nd figure which shows the influence of the density of tau protein on the motion of kinesin in the 1st Embodiment of this invention. It is a conceptual diagram of the gliding assay in the 2nd Embodiment of this invention. It is a conceptual diagram of the method to detect the altered tau protein in the 2nd Embodiment of this invention. It is the microscope picture which image | photographed the microtubule in the 2nd Embodiment of this invention. It is a figure explaining the device used in the experiment in the 2nd Embodiment of this invention. It is a figure which shows the influence of tau protein on the motion of kinesin in the 2nd Embodiment of this invention. It is the figure which compared the motion of the kinesin in the 2nd Embodiment of this invention with 1st Embodiment. It is a figure which shows the influence of incubation time on the motion of kinesin in the 2nd Embodiment of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 2 is a schematic diagram showing microtubules and kinesin in the first embodiment of the present invention, and FIG. 3 is a schematic diagram showing the relationship between microtubules and tau protein in the living body in the first embodiment of the present invention. FIG. 2A is a diagram showing only microtubules and kinesin, FIG. 2B is a diagram showing microtubules and kinesins in nerve cells, and FIG. 3A is a stable microtubule. (B) is a figure which shows the decomposed | disassembled microtubule.

  In the figure, 31 is a microtubule that is a kind of rail molecule in the living body, and 32 is a kind of biomolecular motor that transports a transported substance 36 such as an organelle in a cell such as a nerve cell 51 in the living body. It is kinesin.

  The biomolecular motor is also called a motor protein (Motor Protein), which binds to a cytoskeletal fiber having polarity and moves in a certain direction along the cytoskeletal fiber. There are dozens of types of biomolecular motors in the cell, and the biomolecular motor in the present embodiment may be of any type, such as myosin, dynein, etc. However, since the inventor of the present invention conducted an experiment using the kinesin 32 as a biomolecule motor, the case where the biomolecule motor is the kinesin 32 will be described here.

  In addition, the rail molecule for moving the biomolecular motor is a cytoskeletal fiber in the cell as described above, and may be, for example, an actin filament, but the inventor of the present invention Since the experiment was performed using the microtubule 31 as the rail molecule, here, the case where the rail molecule is the microtubule 31 will be described.

  The microtubule 31 is one of three types of cytoskeletal fibers, and is a cylindrical structure having a diameter of about 25 [nm] and a length of several tens [μm] obtained by polymerizing tubulin as a monomer. A filament comprising: a fiber. Tubulin is a heterodimer in which two globular polypeptides, α-tubulin and β-tubulin, are tightly bound by noncovalent bonds.

  The microtubule 31 has polarity, and one end (the right end in FIG. 2) is a plus end, and the other end (the left end in FIG. 2) is a minus end. The positive end and the negative end are identified by the rate at which tubulin, a subunit constituting the microtubule 31, is polymerized, and the rate at which polymerization is high is called the positive end. The end that slowly extends is called the minus end.

  On the other hand, kinesin 32 is a protein molecule having a head portion of about 10 nm and a total length of about 80 nm. And it has two spherical head parts and an elongated twisted coil part, and the head part alternately repeats the attachment and dissociation to the microtubules 31 so that the fibers can be handled with both hands. Move forward step by step. In this case, the motion of the kinesin 32 consists of 8 [nm] steps, and the maximum speed is about 800 [nm / sec]. As shown by the arrows in FIG. Move toward the positive end. The generative force of kinesin 32 is 5 to 8 [pN].

  MAP called tau protein adheres to the microtubule 31 in the living body, in particular, the microtubule 31 of the cranial nerve cell. As shown in FIG. 3A, normal tau protein, that is, normal tau, contributes to tubulin polymerization and microtubule 31 stability. However, as shown in FIG. 3B, the altered tau protein, that is, the altered tau forms an aggregate and leaves the microtubule 31, so that the microtubule 31 is decomposed.

  In addition, tau protein (tau protein) and normal tau (healthy tau) are synonymous here, and tau protein (protein assembled in the tangles (NFTs) which formed an aggregate with abnormal tau protein (degenerated tau) ) And partially digested or dysfunctional in any way, altered tau protein and altered tau protein tau protein w ) And hyperphosphorylated tau is hyperphosphorylated Tau protein that is not able to bind to MT (tau protein whis is positively phosphorylated and not capturable of binding to MTs), and localized tau is located in a predetermined position. It is synonymous with the known location, and the aggregated tau is synonymous with the tau that has formed an aggregate (tau assembled in tangs sach as NFTs).

  Next, a method for detecting altered tau protein in the present embodiment will be described.

  FIG. 1 is a diagram for explaining a method for detecting altered tau protein using the first detection apparatus according to the first embodiment of the present invention, and FIG. 4 is a diagram of a bead assay according to the first embodiment of the present invention. FIG. 5 is a conceptual diagram, FIG. 5 is a conceptual diagram of a method for detecting altered tau protein in the first embodiment of the present invention, and FIG. 6 is a photomicrograph of the beads in the first embodiment of the present invention. In FIG. 1, (a) to (d) are diagrams showing respective steps, and (c-1), (c-2), (d-1), and (d-2) are (c) and (d). FIG. 5A is a diagram illustrating a state in which kinesin movement is inhibited, and FIG. 5B is a diagram illustrating a state in which kinesin movement is normal.

  The inventor of the present invention uses first and second detection devices described later as a detection device that detects proteins in vitro, that is, in vitro, and a first that uses these first and second detection devices. And a second protein detection method.

  In the present embodiment, a method for detecting the first protein will be described. Here, only a method for detecting altered tau protein will be described as a first protein detection method. Specifically, a method for detecting a protein by observing a bead 36a attached to a protein called “bead assay” (Bead Assay, in vitro Bead Assay) as shown in FIG. By observing the movement state of the kinesin 32 attached with 36a along the microtubule 31 fixed on the base material 22 made of a glass plate or the like, the presence or proportion of the altered tau protein contained in the specimen is determined. Is the method.

In this case, the microtubule 31 is stabilized with a stabilizer. As the stabilizer, taxane-based compounds such as paclitaxel and docetaxel can be used. Here, an example using paclitaxel will be described. That is, an example in which a microtubule (Taxol-stabulated Microtube) 31 stabilized by paclitaxel is used will be described. In such a microtubule 31, the polymerization state of tubulin is stably maintained by paclitaxel and does not contain MAP such as tau protein. Specifically, tubulin collected from pig brain was used in a BRB80 buffer solution (80 [mM] PIPES-NaOH pH = 6.8, 1 containing MgSO ₄ (1 [mM]) and GTP (1 [mM]). [mM] by polymerizing in MgCl ₂ 1 [mM] EGTA) in generating the microtubules 31. After incubating at 37 [° C.] for 30 minutes, the microtubule 31 is stabilized with paclitaxel (40 [μM]) and diluted 100 times in a BRB80 buffer containing paclitaxel (20 [μM]). It was. Note that, depending on the experiment, the microtubule 31 is diluted in a BRB80 buffer containing both tau protein and paclitaxel.

  In the present embodiment, as in the invention described in “Patent Document 1”, a method of fixing the microtubule 31 with the kinesin 32 by inactivating the kinesin 32 by irradiating light of a predetermined wavelength. Adopted. In this method, first, a large number of kinesin 32 molecules are immobilized on the surface of the substrate 22. When the microtubule 31 is introduced here and ATP is given, the microtubule 31 starts to move by the action of the kinesin 32 as a biomolecular motor. That is, since the molecule of kinesin 32 fixed on the surface of the base material 22 moves on the microtubule 31, the microtubule 31 moves on the surface of the base material 22. When the microtubule 31 reaches a desired position, the kinesin 32 is irradiated with light having a predetermined wavelength. As a result, the kinesin 32 is deactivated and stops moving while the microtubule 31 is attached to the head portion of the kinesin 32. Therefore, the microtubule 31 stops. In this case, the head portion of the kinesin 32 is maintained attached to the microtubule 31, so that the microtubule 31 is fixed.

  And in order to observe the movement state of the kinesin 32, the bead 36a coated with the kinesin 32 is used as the transported material 36. Specifically, biotinylated kinesin (GST-410-BCCP kinesin), which has a biotin-dependent enzyme (Biotin-dependent Enzyme) called biotin-carboxyl-carrier-protein (BCCP), was used in the experiment. It was. Biotinylated kinesin was collected from E. coli. After cultivation and purification, the concentration of biotinylated kinesin is 0.07 [mg / ml].

  The beads 36a are commercially available microspheres (CP01N / 6905, manufactured by Bangs Laboratories Inc.) coated with streptavidin and have a diameter of 0.49 [μm]. A specific avidin-biotin binding is used to attach the kinesin 32 molecule to the beads 36a.

  In addition, two types of recombinant tau protein (Tau-441 human 2N4R) are used. One is normal tau protein (T0576, Sigma) and the other is altered Tau protein (P301L, rPeptide).

  As described above, the microtubule 31 stabilized by paclitaxel does not contain MAP. Therefore, when tau protein is attached to the microtubule 31 stabilized by paclitaxel, as shown in FIG. 5 (a), the molecule of kinesin 32 is hindered in its operation, and the operation becomes slow, It is thought that it will leave from 31. However, when tau protein is not attached to the microtubule 31 stabilized by paclitaxel, as shown in FIG. 5B, the operation of kinesin 32 is not hindered, so that the operation is normal. It is considered that it is difficult to detach from the microtubule 31. That is, in vitro, depending on whether tau protein is attached to the microtubule 31 stabilized by paclitaxel, the moving speed and moving distance of the molecule of kinesin 32 moving on the microtubule 31, and the microtubule It is considered that there is a difference in the density (or number) of the molecules of kinesin 32 present on 31.

  On the other hand, if the tau protein is normal, it will adhere to the microtubules 31 stabilized by paclitaxel, but if it has deteriorated to form an aggregate, it will be stabilized by paclitaxel. It is thought that it will not adhere to the formed microtubule 31. That is, when tau protein is supplied to microtubules 31 stabilized by paclitaxel, when the tau protein is normal, the movement of the molecule of kinesin 32 is performed as in FIG. 5 (a). When the tau protein is altered, although it affects the state, that is, the moving speed, the moving distance, and the density (or number), as in the case shown in FIG. It is considered that the movement state is not affected.

  Therefore, if a specimen containing tau protein collected from a subject is supplied to the microtubule 31 stabilized by paclitaxel and the movement state of kinesin 32 is observed, the tau protein contained in the specimen is normal based on the movement state. It is possible to determine whether the protein is an altered or altered protein, and it is also possible to detect the proportion of altered tau protein.

  The inventor of the present invention has invented a method for detecting an abnormal, ie, altered, tau protein based on such findings. In this case, the altered tau protein can be detected using a detection apparatus as shown in FIG.

  In FIG. 1, reference numeral 21 denotes a PDMS (Poly-dimethylsiloxane) film. The PDMS film 21 has one main channel 26 extending linearly and extending in a direction crossing the main channel 26 ( In the example shown, two subchannels 29 are formed which extend orthogonally. One of the subchannels 29 is referred to as a first subchannel 29a, and the other is referred to as a second subchannel 29b. A base material 22 that is a glass slide is attached to the lower surface of the PDMS film 21, and the surface of the base material 22 functions as the bottom surface of the main channel 26 and the subchannel 29.

  Then, as shown in FIG. 1 (b), the microtubule 31 is fixed to the bottom surface of the main channel 26. The microtubule 31 is stabilized by paclitaxel. Subsequently, a solution of tau protein is injected into the subchannel 29. In this case, as shown in FIG. 1C, a normal tau protein solution is injected into the first subchannel 29a, and a modified tau protein solution is injected into the second subchannel 29b. Then, as shown in FIG. 1 (c-1), normal tau protein adheres to the microtubule 31, but as shown in FIG. 1 (c-2), the altered tau protein enters the microtubule 31. Does not adhere.

  Subsequently, as shown in FIG. 1D, beads 36a coated with kinesin 32 are supplied into the main channel 26, and the kinesin 32 is activated. Then, the kinesin 32 moves on the microtubule 31 together with the beads 36a. However, as shown in FIG. 1 (d-1), the operation is inhibited at the place where the normal tau protein is attached. The operation becomes slow or the microtubule 31 is detached. On the other hand, as shown in FIG. 1 (d-2), the kinesin 32 moves smoothly together with the beads 36a because the operation is not hindered at the place where the tau protein is not attached. And it does not detach from the microtubule 31.

  FIG. 6 shows a photomicrograph of the bead 36a coated with kinesin 32 taken by the inventor of the present invention.

  In this way, the state of movement of the kinesin 32 on the microtubule 31 between the location where the normal tau protein was supplied and the location where the altered tau protein was supplied was observed and recorded in advance and collected from the subject. By comparing the movement state of kinesin 32 at the location where the sample containing tau protein is supplied with the recorded result, it becomes possible to determine whether or not the sample contains altered tau protein. It is also possible to determine the proportion of altered tau protein contained.

  Next, in this embodiment mode, a method of an experiment performed by the inventor of the present invention and a structure of a device used in the experiment will be described.

  FIG. 7 is a diagram for explaining a device used in an experiment in the first embodiment of the present invention. In addition, in the figure, (a) is a figure which shows a flow cell, (b) is a schematic diagram of a 1st detection apparatus.

  The inventor of the present invention conducted a basic experiment using a flow cell 10 as shown in FIG. The flow cell 10 includes a pair of cover slips 12 as a transparent plate material made of a glass plate or the like, and a pair of interval holding members 13 for holding an interval between the cover slips 12. The spacing member 13 is made of paperboard impregnated with grease, for example. The bead 36a coated with the microtubule 31 and the kinesin 32 is supplied into a space demarcated by the pair of cover slips 12 and the pair of spacing members 13, and the movement state of the kinesin 32 on the microtubule 31 is changed. Observe.

  In addition, the final first detection apparatus used for determining whether or not the sample contains altered tau protein or for judging the proportion of altered tau protein contained in the sample is shown in FIG. It is the 1st chip | tip 20 as a micro fluidic chip | tip (Micro-Fluidics Chip) as shown in (b). The first chip 20 has a base material 22 that is a glass slide and a PDMS film 21 that adheres to the surface of the base material 22, and the PDMS film 21 extends linearly as described above. One main channel 26 and two subchannels 29 extending so as to be orthogonal to the main channel 26 are formed. One of the subchannels 29 is referred to as a first subchannel 29a, and the other is referred to as a second subchannel 29b. Since the base material 22 is attached to the lower surface of the PDMS film 21, the surface of the base material 22 functions as the bottom surfaces of the main channel 26 and the subchannel 29.

  The PDMS film 21 is produced by using a photolithography technique in the same manner as the invention described in “Patent Document 1”. For example, using SU-850, which is a resist commercially available from MicroChem, a surface of a silicon wafer (not shown) having a shape corresponding to the main channel 26 and the subchannel 29 and having a height of about 50 [μm]. A convex pattern is formed. Next, a PDMS prepolymer such as Silgard 184 commercially available from Dow Corning, for example, is applied to the surface of the silicon wafer, the prepolymer is cured, and then peeled off. Thereby, the PDMS film 21 as shown in FIG. 7B can be obtained.

  Finally, after the PDMS film 21 is exposed to oxygen plasma (50 [sccm], 20 [Pa], 65 [W], 8 [sec]), the PDMS film 21 is brought into close contact with the surface of the substrate 22. Join.

  In the first chip 20 created by the inventor of the present invention, the depth, width and length of the main channel 26 are 50 [μm], 200 [μm] and 20 [mm], respectively. The depth, width and length of the subchannel 29 are 50 [μm], 100 [μm] and 15 [mm], respectively.

  Furthermore, the inventors of the present invention used an inverted microscope (Olympus IX-71) equipped with a DIC (Differential Interference Contrast) setup for observation of the experiment. Then, an image was recorded as experimental data using a photometrics camera (Cascade 512II), and the recorded experimental data was processed using the software MetaMorph and Cosmos.

  The flow cell 10 was directly held on the stage of an inverted microscope. Further, as shown in FIG. 7B, two syringe pumps 41 made by Kd Scientific were connected to the first chip 20. Each syringe pump 41 is connected to the outlet 26out of the main channel 26 and the outlet 29out of the subchannel 29 via the connection pipe 42, respectively, and is used to suck out the liquid in the main channel 26 and the subchannel 29. . In FIG. 7B, 26 in and 29 in are entrances of the main channel 26 and the sub channel 29.

  In order to detect the activity of the kinesin 32, it is necessary to observe the movement of the kinesin 32 along the microtubule 31. Therefore, as described above, by using the bead 36a coated with kinesin 32, the movement state of the kinesin 32 along the microtubule 31 fixed by using the DIC setup, typically, the average moving speed is obtained. It becomes possible to measure.

  In the experiment, a cover slip 12 composed of a glass plate coated with PLL (Poly-L-Lysine), which is a polycation capable of binding to a negatively charged protein, and a base material 22 are used for fixing the microtubule 31. did. There are two methods for preparing the microtubule 31 to which the tau protein is attached.

  In the first method, microtubules 31 to which tau protein is attached (TMT: microtubule 31 to which normal tau protein is supplied or MuTMT: microtubule 31 to which altered tau protein is supplied) are prepared separately. Is the method. In this case, the microtubule 31 diluted 100-fold with a BRB80 buffer containing normal tau protein or a BRB80 buffer containing altered tau protein is incubated at 37 [° C.] for 30 minutes in the tube. Then, the microtubule 31 to which the tau protein is adhered is injected into the flow cell 10 coated with PLL and incubated for 3 minutes. After flowing the beads 36a coated with kinesin 32, 1 [mM] ATP solution is poured to activate kinesin 32.

  The second method is an on-site deposition. In this case, the microtubule 31 diluted 100 times in the BRB80 buffer is first incubated for 3 minutes to be immobilized in the flow cell 10 coated with PLL. Then, in order to attach the tau protein to the microtubule 31, a tau protein solution is injected. Subsequently, as described later, incubation for attaching the tau protein to the microtubule 31 is performed for different times. The microtubule 31 (TMT) to which the tau protein is attached is used to observe the operation of the bead 36a coated with kinesin 32 in the presence of 1 [mM] ATP solution.

  In the second method, i.e. on-site deposition, the first detector as shown in Fig. 1 was also used. The main channel 26 is first coated with PLL, and the microtubule 31 is fixed in the main channel 26. A solution of tau protein is injected into one subchannel 29 and, as a control experiment, BRB80 buffer is injected into the other subchannel 29. After incubating for 25 minutes, beads 36 a coated with kinesin 32 are poured into the main channel 26. Finally, 1 [mM] ATP solution is added to activate the kinesin 32 attached to the beads 36a.

  Next, the contents and results of the experiment conducted by the inventor of the present invention will be described.

  FIG. 8 is a diagram showing the effect of tau protein on kinesin movement in the first embodiment of the present invention, and FIG. 9 is the effect of tau protein density on kinesin movement in the first embodiment of the present invention. FIG. 10 is a diagram showing the effect of incubation time on the movement of kinesin in the first embodiment of the present invention, and FIG. 11 is the effect on the number of molecules of kinesin in the first embodiment of the present invention. FIG. 12 is a diagram showing the influence of incubation time, FIG. 12 is a diagram showing the influence of the state of microtubules on the movement of kinesin in the first embodiment of the present invention, and FIG. 13 is kinesin in the first embodiment of the present invention. It is a 2nd figure which shows the influence of the density | concentration of the tau protein which acts on movement.

  The inventor of the present invention conducted experiments under three different conditions.

  The first condition corresponds to the first method described above, in which tau protein is attached to the microtubule 31 in advance before fixing the microtubule 31 in the flow cell 10. A tubulin monomer: tau protein molecule ratio of 40: 1 was used, and microtubules 31 diluted 100-fold with a BRB 80 buffer containing normal tau protein or altered tau protein were treated at 20 ° C. at 20 ° C. Incubated for minutes. As a control experiment, normal microtubules 31 were diluted 100 times in a BRB80 buffer and incubated at 37 [° C.] for 20 minutes. After performing the above-described predetermined experimental procedure, the beads 36a coated with kinesin 32 were observed, and the average moving speed in each case was measured.

  The measured value of the average moving speed of the beads 36a along the MT which is the normal microtubule 31 and the average moving speed of the beads 36a along the MuTMT which is the microtubule 31 to which the altered tau protein is attached are respectively shown. They were 0.22 ± 0.06 [μm / sec] (n = 45) and 0.22 ± 0.04 [μm / sec] (n = 44). Note that n is the number of observed beads 36a. Moreover, the measured value of the average moving speed of the beads 36a along the TMT, which is the microtubule 31 to which the normal tau protein was attached, was 0.19 ± 0.05 [μm / sec] (n = 45). .

  As shown in FIG. 8, the meaning is clear by comparing the obtained results with the control experiment. FIG. 8 shows a microtubule in which a tau protein of a kind different from the case where the normalized average moving speed of the bead 36a coated with kinesin 32 in the flow cell 10 moves along the normal microtubule 31 is attached. It shows in comparison with the case of moving along 31. In FIG. 8, MT means a normal microtubule 31, MuTMT means a microtubule 31 supplied with a modified tau protein, and TMT means a microtubule 31 supplied with a normal tau protein. The error bar corresponds to the standard deviation.

  In addition, in the result of Student's t test (for example, see Non-Patent Document 4), the values of the case where the altered tau protein was attached and the case where the normal tau protein was attached were respectively p = 0. .86 and p <0.01.

S.M. Dunn, A. Constantinides, P.V.Moghe, “Numerical Methods in Biomedical Engineering”, Elsevier Academic Press, 2006

  The inventors of the present invention also conducted experiments on the influence of the density of tau protein on the average moving speed of the beads 36a. FIG. 9 shows the change in the normalized average moving speed of the beads 36a coated with kinesin 32 in the flow cell 10 when the ratio between the microtubule 31 and the tau protein is changed. As shown in FIG. 9, when the ratio of the number of microtubules 31 to the number of tau proteins is changed, the normalized average moving speed of the beads 36a along the microtubules 31 is the number of microtubule monomers. And the number of tau protein molecules are 8: 1, 80: 1, 800: 1 and the number of tau proteins is 0, 0.11 ± 0.02 [μm / sec] (n = 32), 0.12 ± 0.03 [μm / sec] (n = 44), 0.13 ± 0.02 [μm / sec] (n = 24), and 0.14 ± 0.02 [ μm / sec] (n = 45). Thus, the result that the average moving speed of the bead 36a falls, so that the tau protein adhering to the microtubule 31 increased was obtained.

  The second condition corresponds to the second method described above, and the tau protein is attached to the microtubule 31 in the flow cell 10 on-site. In this experiment, after fixing the microtubule 31, a 1 μg / ml tau protein solution was injected into the flow cell 10. And the average moving speed of the bead 36a coated with kinesin 32 was measured every different incubation time (0 minutes, 5 minutes, 20 minutes, 60 minutes and 120 minutes).

  As shown in FIG. 10, the result that the average moving speed of the beads 36a decreased with the increase of the incubation time was obtained. FIG. 10 shows the normalized average migration rate of the beads 36a coated with kinesin 32 when the tau protein is attached to the microtubule 31 in the flow cell 10 in-situ at different incubation times. It shows. The numbers n of the beads 36a observed were n = 65, 44, 25, 20, and 8, respectively. In FIG. 10, error bars correspond to the standard deviation, and the X-axis scale is not given at a constant rate.

  Further, FIG. 11 shows the observed average number of moving beads 36a. Thereby, the influence in the case of attaching tau protein to the microtubule 31 fixed in the flow cell 10 on-site can be understood. In FIG. 11, error bars correspond to standard deviations, and the scale of the X axis is not given at a constant rate.

  Note that the number of moving beads 36a observed in the control experiment (incubation time = 0 minutes) was 17 or more per observation. However, as the incubation time increased, the number of moving beads 36a decreased and was 2 per observation at the longest incubation time (incubation time = 120 minutes). Four observations were made at each incubation time.

  The third condition corresponds to the second method described above, and attaches tau protein on-site to the microtubule 31 in the first detection apparatus as shown in FIG. . As described above, the main channel 26 was coated with PLL, and the microtubule 31 was fixed. Then, 1 [μg / ml] tau protein solution was injected into one subchannel 29 and incubated for 25 minutes. As a control experiment, BRB80 buffer was injected into the other subchannel 29. FIG. 12 shows the result of the preliminary experiment. The measured value of the average moving speed of the beads 36a coated with kinesin 32 on the normal microtubule 31 is 0.32 ± 0.08 [μm / sec] (n = 15), and normal tau protein is attached. The measured value of the average moving speed of the beads 36a coated with kinesin 32 on the microtubule 31 was 0.24 ± 0.03 [μm / sec] (n = 9).

  FIG. 12 shows a comparison of the normalized average moving speed of the beads 36a coated with kinesin 32 at the intersection of the main channel 26 and the different subchannel 29. Note that the numbers n of the observed beads 36a were n = 15 and 9, respectively. Compared with the intersection with the subchannel 29 into which only the BRB80 buffer solution was injected, the average movement speed was greatly reduced at the intersection with the subchannel 29 into which the tau protein solution was injected. Note that the error bars in FIG. 12 correspond to the standard deviation.

  Although tau protein is known to play an important role in the stabilization of microtubules 31, these experimental results indicate that tau protein has a negative effect on kinesin 32 movement. This is because the stabilization of the microtubule 31 is achieved by paclitaxel. This indicates that tau protein is not beneficial for the stabilization of microtubules 31 but acts as a resistance to kinesin 32. This finding is consistent with the experimental results shown in FIG.

  Normal tau protein adheres to the microtubule 31. In contrast, altered tau proteins tend to bind together to form aggregates. And if it becomes such an aggregate, the binding ability to the microtubule 31 will fall (for example, refer nonpatent literature 1). From this, it can be expected that when TMT, which is a microtubule 31 supplied with normal tau protein, is used, the average moving speed of the beads 36a coated with kinesin 32 is lowered. Furthermore, since tau protein does not adhere to the microtubule 31, the kinesin 32 is used when the MuTMT, which is the microtubule 31 to which the altered tau protein is supplied, and when the MT, which is the normal microtubule 31, is used. It can be expected that there will be no difference in the average moving speed of the coated beads 36a.

  Using a device as described above as a molecular detector requires the ability to provide information about tau protein attachment levels. FIG. 10 shows that as the incubation time increases, the amount of tau protein attached to the microtubule 31 increases, resulting in a decrease in the average migration rate of the beads 36a coated with kinesin 32. Further, when the amount of tau protein attached increases, the operation of kinesin 32 (and the adhesion of kinesin 32 to microtubule 31 is also inhibited), so that beads 36a coated with kinesin 32 become microtubules. It will detach easily from 31. Then, as shown in FIG. 11, the number of beads 36a coated with kinesin 32 that moves along TMT, which is a microtubule 31 supplied with normal tau protein, decreases as the incubation time increases.

  The inventor of the present invention also conducted experiments on the influence of the density of altered tau protein on the average migration speed of the beads 36a coated with kinesin 32. FIG. 13 shows the normalized average moving speed of the beads 36a coated with kinesin 32 in the flow cell 10 when the density of the normal tau protein and the density of the tau protein that has changed into aggregates are changed. Shows changes. In the example shown in FIG. 13, in cases A to E, the number of normal tau proteins relative to the number of microtubules 31, the number of tau proteins that became aggregates relative to the number of microtubules 31, and the value of n are {10 times, 1 time, n = 24}, {10 times, 0, n = 33}, {11 times, 0, n = 20}, {10 times, 10 times, n = 35}, and {0, 10 times, n = 27}. Thus, when using MuTMT, which is a microtubule 31 supplied with altered tau protein, kinesin 32 is coated more than when using TMT, which is a microtubule 31 supplied with normal tau protein. As a result, the average moving speed of the beads 36a was high.

  The use of the first chip 20 as shown in FIG. 7B is important in order to suppress errors caused by the experimental configuration as much as possible because it can be studied under different conditions. When the flow cell 10 is used, it is necessary to prepare different apparatuses according to individual experiments. On the other hand, the first chip 20 is an excellent experimental device because the experiment can be performed under exactly the same conditions. This is because tau protein molecules can be attached to different portions of the same microtubule 31 at different locations of the main channel 26. Further, beads 36a coated with the same kinesin 32 can be moved through the main channel 26 for different cases. The ability to provide the same conditions for comparing different cases is an important advantage for the experimental apparatus in order to obtain stable results.

  As described above, in the present embodiment, the microtubule 31 is stabilized by paclitaxel, the microtubule 31 is attached to the base material 22, a specimen containing tau protein is supplied to the microtubule 31, and the microtubule 31 is moved over the microtubule 31. Based on the movement state of the moving kinesin 32, the ratio of the altered tau protein contained in the specimen is detected.

  The first chip 20 extends in a direction intersecting the main channel 26 with the main channel 26 having the bottom surface of the base material 22 to which the microtubules 31 stabilized by paclitaxel are attached, and the tau protein. The moving state of the kinesin 32 that moves on the microtubule 31 can be observed.

  This makes it possible to easily and reliably detect the proportion of altered tau protein contained in the specimen in vitro. Therefore, it is possible to diagnose abnormal tau protein diseases such as Alzheimer's disease at a stage where the ratio of altered tau protein is low. For example, it functions as a normal tau protein using a phosphorylase inhibitor or the like. By recovering, early treatment becomes possible.

  Next, a second embodiment of the present invention will be described. In addition, about the thing which has the same structure as 1st Embodiment, the description is abbreviate | omitted by providing the same code | symbol. The description of the same operation and the same effect as those of the first embodiment is also omitted.

  FIG. 14 is a conceptual diagram of a gliding assay in the second embodiment of the present invention, FIG. 15 is a conceptual diagram of a method for detecting altered tau protein in the second embodiment of the present invention, and FIG. It is the microscope picture which image | photographed the microtubule in 2nd Embodiment. In FIG. 15, (a) shows a state in which kinesin movement is inhibited, and (b) shows a state in which kinesin movement is normal.

  In the present embodiment, a method for detecting the second protein will be described. Note that description of matters similar to those in the first embodiment is omitted.

  Here, only a method for detecting altered tau protein will be described as a method for detecting the second protein. Specifically, it is a method for detecting a protein by observing a microtubule 31 called a gliding assay (Gliding Assay, in vitro gliding assay) as shown in FIG. Using the fluorescent microtubule, the altered tau protein contained in the specimen is observed by observing the movement state of the fluorescent microtubule along the surface of the base material 22 made of a glass plate coated with kinesin 32 or the like. It is a method to judge the existence and the ratio of. As in the invention described in “Patent Document 1”, the fluorescent microtubule is a mixture of fluorescent tubulin labeled with a fluorescent dye and fluorescent tubulin not labeled with a fluorescent dye in an appropriate ratio. Can be obtained.

  The microtubule 31 is stabilized by paclitaxel, as in the first embodiment. Therefore, when tau protein is attached to the microtubule 31, as shown in FIG. 15A, the kinesin 32 is hindered by the tau protein, and the kinesin 32 is slowed down. It is thought that it will leave. However, when the tau protein is not attached to the microtubule 31, as shown in FIG. 15 (b), the kinesin 32 has a normal operation because the operation is not inhibited by the tau protein. It is considered that kinesin 32 is not easily detached from the surface of the base material 22. That is, in vitro, depending on whether or not tau protein is attached to the microtubule 31 stabilized by paclitaxel, the moving speed and moving distance of the microtubule 31 moving on the base material 22 coated with kinesin 32 In addition, it is considered that a difference occurs in the density (or number) of the microtubules 31 existing on the base material 22.

  Therefore, if a specimen containing tau protein collected from a subject is supplied to the microtubule 31 stabilized by paclitaxel and the movement state of the microtubule 31 is observed, the tau protein contained in the specimen based on the movement state It is possible to determine whether the protein is normal or altered, and it is also possible to detect the proportion of altered tau protein.

  Next, in this embodiment mode, a method of an experiment performed by the inventor of the present invention and a structure of a device used in the experiment will be described.

  FIG. 17 is a diagram for explaining a device used in an experiment in the second embodiment of the present invention. In the figure, (a) is a schematic diagram of the first detection device, (b) is a photograph showing a state where a microtubule enters the nanochannel, and (c) is a photograph showing the created nanochannel.

  In the figure, reference numeral 30 denotes a second chip which is a microfluidic chip created by the inventor of the present invention as a second detection device. The second chip 30 includes a nanochannel part 35, inlet parts 37 connected to both ends of the nanochannel part 35, and a fluorescence detection part 38 as an outlet part.

  Similar to the nanochannel in the invention described in “Patent Document 1”, the nanochannel portion 35 is formed by using a photolithography technique, and a glass plate and a PDMS film bonded to the glass plate. And having a plurality of parallel elongated grooves, that is, nanochannels, formed in the PDMS film as shown in FIG. 17 (c). Therefore, the bottom surface of the nanochannel is formed by the surface of the glass plate. The surface of the glass plate is coated with kinesin 32.

  The inlet portion 37 is a space connected to one end of the nanochannel portion 35, and is a portion to which the microtubule 31 diluted in the BRB80 buffer is supplied. The microtubule 31 supplied to the inlet portion 37 is conveyed by the kinesin 32 fixed to the upper surface of the glass plate, and probabilistically enters the nanochannel as shown in FIG. Since kinesin 32 moves on the microtubule 31 from the minus end side toward the plus end side, the microtubule 31 moves in the direction indicated by the arrow.

  The fluorescence detection unit 38 is a space connected to one end of the nanochannel unit 35 and is a unit for observing the microtubule 31 carried out from the nanochannel unit 35. As described above, depending on whether or not tau protein is attached to the microtubule 31 stabilized by paclitaxel, the moving speed and moving distance, and the density (or number) of the microtubule 31 transported by the kinesin 32 are determined. Since the difference occurs, if the length of the nanochannel portion 35 and the density (or number) of the microtubules 31 supplied to the inlet portion 37 are given as given conditions, the microtubules 31 are introduced into the inlet portion 37. After a certain period of time has passed since the supply, the number of microtubules 31 present in the fluorescence detection unit 38 is counted, whereby the moving speed and moving distance of the microtubules 31 transported by the kinesin 32, and the density (Or number) can be measured. Since the fluorescence intensity in the fluorescence detection unit 38 is considered to be proportional to the number of microtubules 31 present therein, by detecting the fluorescence intensity in the fluorescence detection unit 38, the moving speed and the moving distance of the microtubes 31, In addition, the density (or number) may be measured.

  Next, the contents and results of the experiment conducted by the inventor of the present invention will be described.

  FIG. 18 is a diagram showing the effect of tau protein on the movement of kinesin in the second embodiment of the present invention, and FIG. 19 shows the movement of kinesin in the second embodiment of the present invention as compared with the first embodiment. FIG. 20 and FIG. 20 are diagrams showing the influence of the incubation time on the movement of kinesin in the second embodiment of the present invention.

  FIG. 18 shows a case where the average moving speed of the microtubule 31 passing through the nanochannel portion 35 of the second chip 30 is different from that of the normal microtubule 31 in the case of the microtubule 31 to which a different type of tau protein is attached. Compare and show. In FIG. 18, MT is a normal microtubule 31, which is incubated for 20 minutes at 37 [° C.] in a BRB80 buffer, and MuTMT is a microtubule 31 to which a modified tau protein is attached, Incubated for 20 minutes at 37 [° C.] in a BRB80 buffer containing a modified tau protein, TMT is a microtubule 31 with a normal tau protein attached, and in a BRB80 buffer containing a normal tau protein Incubated at 37 [° C.] for 20 minutes, and MuTTMT is a microtubule 31 to which a normal tau protein and a modified tau protein are attached, and is 37 [in a BRB80 buffer containing normal tau protein. At 20 ° C.] and BRB80 buffer containing altered tau protein In is obtained by incubation at 37 [℃] 20 minutes.

  The measured values of the average moving speed of the microtubule 31 transported by the kinesin 32 are 0.289 ± 0.036 [μm / sec] (n = 60), 0, 0 for MT, MuTMT, TMT, and MuTTMT, respectively. 272 ± 0.026 [μm / sec] (n = 60), 0.230 ± 0.034 [μm / sec] (n = 60), and 0.184 ± 0.023 [μm / sec] (n = 60). Note that n is the number of microtubules 31 observed.

  A value obtained by normalizing the average moving speed of the microtubule 31 obtained by a method called a gliding assay as described in the present embodiment is referred to as a bead assay as described in the first embodiment. When the average moving speed of the beads 36a obtained by the method described above is compared with the normalized value, it is as shown in FIG. In FIG. 19, □ indicates a value in the case of a gliding assay, and ♦ indicates a value in the case of a bead assay.

  In the example shown in FIG. 19, the ratio between the number of microtubules 31 and the number of tau proteins is 80: 1. Furthermore, the ratio of MT, MuTMT, TMT and MuTTMT are 1: 0.94: 0.80: 0.64 and 1: 0.99: 0.75: 0 for the gliding assay and bead assay, respectively. .64.

  FIG. 20 shows the relationship between the incubation time and the average moving speed of the microtubule 31. Note that □ indicates a value when the microtubule 31 is MT, and ◆ indicates a value when the microtubule 31 is TMT. It can be seen that when the microtubule 31 is TMT, the average moving speed of the microtubule 31 decreases as the incubation time increases.

  As described above, in this embodiment, the microtubule 31 is stabilized by paclitaxel, the molecule of kinesin 32 is attached onto the base material 22, the sample containing tau protein is supplied to the microtubule 31, and is moved by the kinesin 32. Based on the movement state of the microtubule 31 to be caused, the ratio of the altered tau protein contained in the specimen is detected.

  The second chip 30 is connected to a nanochannel part 35 including a plurality of nanochannels whose bottom surface is the surface of the base material 22 to which the kinesin 32 is attached, and is connected to one end of the nanochannel part 35, and is stabilized by paclitaxel. Inlet part 37 to which microtubule 31 is supplied and fluorescence detection part 38 connected to the other end of nanochannel part 35, supplied to inlet part 37, passed through nanochannel part 35 and discharged A fluorescence detection unit 38 capable of observing the microtubule 31.

  This makes it possible to easily and reliably detect the proportion of altered tau protein contained in the specimen in vitro. Therefore, it is possible to diagnose abnormal tau protein diseases such as Alzheimer's disease at a stage where the ratio of altered tau protein is low. For example, it functions as a normal tau protein using a phosphorylase inhibitor or the like. By recovering, early treatment becomes possible.

  In addition, this invention is not limited to the said embodiment, It can change variously based on the meaning of this invention, and does not exclude them from the scope of the present invention.

  The present invention can be used in a protein detection method and a detection apparatus.

22 Substrate 26 Main channel 29a First subchannel 29b Second subchannel 35 Nanochannel part 36a Bead 37 Inlet part 38 Fluorescence detection part

Claims (11)

A method for detecting a protein comprising a polarity and a rail molecule in which a biomolecule motor moves in a direction corresponding to the polarity, and a relative movement state of the biomolecule motor moving on the rail molecule,
(A) stabilizing the rail molecule with a stabilizer,
(B) either one of the rail molecule or the biomolecule motor is attached on a substrate;
(C) supplying a sample containing tau protein to the rail molecule;
(D) detecting the ratio of the altered tau protein contained in the specimen based on the movement state of the biomolecule motor moving on the rail molecule or the movement state of the rail molecule moved by the biomolecule motor. A method for detecting a characteristic protein. Based on the fact that the movement of the biomolecular motor on the rail molecule stabilized by the stabilizer is inhibited by the normal tau protein contained in the specimen and not by the altered tau protein contained in the specimen. The method for detecting a protein according to claim 1, wherein the ratio of the altered tau protein contained in the specimen is detected. The protein detection method according to claim 1, wherein the stabilizer is paclitaxel or docetaxel. The moving state is the moving speed, moving distance, density or number of the biomolecule motor moving on the rail molecule, or the moving speed, moving distance, density, or number of the rail molecule moved by the biomolecule motor. The method for detecting a protein according to claim 2. Claims: A microsphere having a biomolecule motor joined to rail molecules attached on a substrate is supplied, and the movement state of the biomolecule motor moving on the rail molecule is determined based on the movement state of the microsphere. Item 5. A method for detecting a protein according to Item 4. 5. A rail molecule having fluorescence is supplied to a biomolecule motor attached on a base material, and the movement state of the rail molecule moved by the biomolecule motor is determined based on the movement state of the rail molecule. The method for detecting a protein according to 1. It is used in a protein detection method that is based on a relative movement state between a rail molecule having a polarity and a biomolecule motor moving in a direction corresponding to the polarity and a biomolecule motor moving on the rail molecule. A detection device,
(A) a main channel whose bottom surface is the surface of a base material to which rail molecules stabilized by a stabilizer are attached;
(B) extending in a direction crossing the main channel and having a subchannel to which a specimen containing tau protein is supplied;
(C) A detection device characterized in that a movement state of a biomolecule motor moving on the rail molecule can be observed. The detection apparatus according to claim 7, wherein the subchannel is a plurality, and the influence of the tau protein contained in the plurality of samples on the movement state of the biomolecule motor moving on the rail molecule can be simultaneously observed. 8. The movement state of a biomolecule motor that moves on the rail molecule is determined based on a movement state of the microsphere, wherein a microsphere having a biomolecule motor bonded thereto is supplied to the main channel. Detection device. It is used in a protein detection method that is based on a relative movement state between a rail molecule having a polarity and a biomolecule motor moving in a direction corresponding to the polarity and a biomolecule motor moving on the rail molecule. A detection device,
(A) a nanochannel portion including a plurality of nanochannels whose bottom surface is the surface of a base material to which the biomolecule motor is attached;
(B) an inlet portion connected to one end of the nanochannel portion and supplied with rail molecules stabilized by a stabilizer;
(C) an outlet portion connected to the other end of the nanochannel portion, the outlet portion being supplied to the inlet portion and capable of observing rail molecules discharged through the nanochannel portion. A detection device characterized by. The detection apparatus according to claim 10, wherein rail molecules supplied to the inlet portion have fluorescence, and the outlet portion can detect fluorescence intensity.